The overall goal of this application is to continue our work funded by a MERIT award on the dynamics and evolution of a bacterial group II intron. Group I and group II introns are self-splicing elements with a wide phylogenetic distribution, reflecting their dispersal through different mobility mechanisms. These introns initiate their own movement, and then tap into established pathways of recombination and/or repair to arrive at new destinations, in either familiar or foreign genetic neighborhoods. Group II introns are particularly important elements because they are the putative progenitors of eukaryotic spliceosomal introns, and they appear to be ancestrally related to mammalian retroelements, which, like group II introns, transpose via an RNA intermediate. Bacterial group II introns therefore provide an excellent model system for the function of these prevalent eukaryotic elements, which together occupy >50% of the human genome. Of additional importance is that site-specific DNA integration of group II introns is being exploited for gene targeting in biomedical applications. Our three specific aims all involve host-retroelement relationships and each is based on significant discoveries made during the previous funding period, as follows: First, we will further explore our findings that dissemination of the Lactococcus lactis group II intron, Ll.LtrB, is highly sensitive to host cell starvation, involving a dynamic interplay with small-molecule effectors ppGpp and cAMP. We are building high-throughput analytic tools to explore this relationship between the group II intron and the host's intra- and extracellular environment, and to test the hypothesis that the ribosome is the node of cellular networks that regulate retrotransposition. Second, we will pursue another recent discovery, namely the role in retrotransposition of the conjugative plasmid pRS01, on which the Ll.LtrB intron resides. Additionally, based on evidence that group II introns are subject to horizontal gene transfer, we will ask whether the intron RNA itself might be the transfer agent. Third, we will investigate the provocative finding that nuclear expression of the Ll.LtrB intron prevents the mature transcript from being expressed in yeast. This silencing of the transcript may have provided the selective pressure for the evolution of spliceosomal introns from group II introns. We will use genetic methodologies to probe host surveillance of group II introns and, potentially, gain further insight into evolution of a group II intron to spliceosome dependence. Thus, using classical and high-throughput genetic and biochemical techniques in bacteria and yeast, we will build on our discoveries, to explore the lifestyle, host inter-dependence and evolution of group II introns. The impact of these studies is an enhanced understanding of the function and evolution of these elements. Additionally, the work will facilitate the exploitation of group II introns as agents of DNA manipulation and gene therapy.